Nanoliter array loading

ABSTRACT

An interface is provided for storing microfluidic samples in a nanoliter sample chip. A fluid access structure provides a fluid access region to a selected subset of sample wells from an array of sample wells. A fluid introduction mechanism introduces a sample fluid to the fluid access region so that the sample wells in the selected subset are populated with the sample fluid without the unselected sample wells being populated with the sample fluid.

This application claims priority from U.S. Provisional PatentApplication 60/552,267, filed Mar. 11, 2004; U.S. Provisional PatentApplication 60/607,838, filed Sep. 8, 2004; and U.S. Provisional PatentApplication 60/627,334, filed Nov. 12, 2004; all of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention generally relates to techniques for assaying sampleliquids, and more specifically to techniques for utilizing a sub-set ofnanoliter sample volumes in an array.

Various systems are known for performing a large number of chemical andbiological storage assays and synthesis operations. One approach uses anassay chip having an array of nanoliter volume through-hole sample wellswith hydrophilic interiors and openings surrounded by hydrophobicmaterial. One specific commercial example of a nanoliter chip system isthe Living Chip™ made by BioTrove, Inc. of Woburn, Mass. Nanoliter chiptechnology relies on the ability to handle very small volumes of fluidsamples, typically, 100 nanoliters or less. The various considerationstaken into account in handling such small liquid samples are known asmicrofluidics.

FIG. 1 shows a cut away view of a typical nanoliter sample chip. This isdescribed, for example, in U.S. Pat. No. 6,387,331 and U.S. PatentApplication 20020094533, the contents of which are incorporated hereinby reference. Array chip 10 contains an array of through-hole samplewells 12 that traverse the chip 10 from one planar surface 14 to theother opposing planar surface (not shown).

The sample wells 12 may be grouped into sub-arrays such as bycontrolling the spacing between the wells. For example, FIG. 2 shows achip 10 in which the sample wells 12 are grouped into a 4 by 12 array of5-well by 5-well sub-arrays 20. In another embodiment, the sub-arrays 20may be 8-wells by 8-wells or any other convenient number. The chip 10 inFIG. 2 is 1″×3″ to correspond to a standard microscope slide. The samplewells 12 in a sub-array 20 may be laid out in a square or rectangulargrid arrangement as shown in FIG. 2, or the rows and/or columns ofsample wells may be offset as shown in FIG. 1.

The sample chip 10 typically may be from 0.1 mm to more than 10 mmthick; for example, around 0.3 to 1.52 mm thick, and commonly 0.5 mm.Typical volumes of the through-hole sample wells 12 could be from 0.1picoliter to 1 microliter, with common volumes in the range of 0.2-100nanoliters, for example, about 35 nanoliters. Capillary action orsurface tension of the liquid samples may be used to load the samplewells 12. For typical chip dimensions, capillary forces are strongenough to hold liquids in place. Chips loaded with sample solutions canbe waved around in the air, and even centrifuged at moderate speedswithout displacing samples.

To enhance the drawing power of the sample wells 12, the target area ofthe receptacle, interior walls 42, may have a hydrophilic surface thatattracts a sample fluid. It is often desirable that the surfaces bebio-compatible and not irreversibly bind biomolecules such as proteinsand nucleic acids, although binding may be useful for some processessuch as purification and/or archiving of samples. Alternatively, thesample wells 12 may contain a porous hydrophilic material that attractsa sample fluid. To prevent cross-contamination (crosstalk), the exteriorplanar surfaces 14 of chip 10 and a layer of material 40 around theopenings of sample wells 12 may be of a hydrophobic material such as amonolayer of octadecyltrichloroeilane (OTS). Thus, each sample well 12has an interior hydrophilic region bounded at either end by ahydrophobic region.

The through-hole design of the sample wells 12 avoids problems oftrapped air inherent in other microplate structures. This approachtogether with hydrophobic and hydrophilic patterning enable self-meteredloading of the sample wells 12. The self-loading functionality helps inthe manufacture of arrays with pre-loaded reagents, and also in that thearrays will fill themselves when contacted with an aqueous samplematerial.

It has been suggested that such nanoliter chips can be utilized formassively parallel assays such as Polymerase Chain Reaction (PCR) andEnzyme-Linked Immunosorbent Assay (ELISA) analysis. However, one problemwith such applications of nanoliter chips is the complex time-consumingpreparation and processing of the chip that is required. Before thesamples are introduced, each sample Well must be pre-formatted with thenecessary probes, reagents, etc. which will be referred to generally asreagents. Such chip preparation wilt be referred to as formatting. Oncethe chip is formatted, the analyte or specimen must be introduced intoeach well, which will be referred to as sample loading. The term samplewill be used to refer generically to both specimens and reagents.Transferring of large collections of fluid samples such as libraries ofsmall molecule drug candidates, cells, probe molecules (e.g.,oligomers), and/or tissue samples stored in older style 96- or 384-wellplates into more efficient high density arrays of nanoliter receptaclescan be difficult. As a practical matter, there tend to be two approachesto formatting and loading of nanoliter sample chips—bulk transfer anddiscrete transfer.

An example of bulk transfer is dipping a sample chip into a reservoir ofsample liquid. The sample liquid wicks into the sample wells bycapillary action and all of the wells fill uniformly with the sample.

One established method for discrete transfer uses a transfer pin loadedwith the transfer liquid. For example, pins or arrays of pins aretypically used to spot DNA samples onto glass slides for hybridizationanalysis. Pins have also been used to transfer liquids such as drugcandidates between microplates or onto gels (one such gel system isbeing developed by Discovery Partners, San Diego, Calif.). Many pintypes are commercially available, of various geometries and deliveryvolumes. V&P Scientific of San Diego, Calif. makes slotted, grooved,cross-hatched, and other novel-geometry pink. The Stealth Pin by ArrayItis capable of delivering hundreds of spots in succession from one sampleuptake, with delivery volumes of 0.5 nL to 2.5 nL. Major PrecisionEngineering sells pins having tapered tips and slots such as theMicroQuil 2000. Techniques for using a one or more pins to transfersample liquid are described in U.S. patent application Ser. No.10/227,179, filed Aug. 23, 2002, and incorporated herein by reference.

SUMMARY OF THE INVENTION

Representative embodiments of the present invention include methods andsystems for providing an interface for storing microfluidic samples in ananoliter sample chip. A fluid access structure provides a fluid accessregion to a selected subset of sample wells from an array of samplewells. A fluid introduction mechanism introduces a sample fluid to thefluid access region so that the sample wells in the selected subset arepopulated with the sample fluid without the unselected sample wellsbeing populated with the sample fluid.

In further embodiments, the fluid access structure may be adapted forpositioning next to a planar surface of the array to provide the fluidaccess region. The fluid access structure may include at least onemicrofluidic circuit for distributing the sample fluid to the fluidaccess region, which may be fixed to the array or detachable from thearray.

In another embodiment, the fluid access structure may be adapted to folda portion of the array to provide the fluid access region. For example,the fluid access structure may be adapted to fit into a microplatesample well so as to enable introducing a sample fluid within themicroplate sample well into the fluid access region.

In other embodiments, the fluid access structure may include a mask tocreate a barrier between the fluid access region and the rest of thearray. Or a printing plate may be used as the fluid access structure andthe fluid introduction mechanism. The fluid introduction mechanism maybe based on dragging a drop of the sample fluid over the fluid accessregion. Or the fluid introduction mechanism may be adapted fordispensing a focused drop of the sample fluid into the fluid accessregion, such as by spraying. In various embodiments, a sponge or apipette may be used for the fluid introduction mechanism.

In another embodiment, a membrane is used as the fluid access structureand fluid introduction mechanism. The membrane may include an outersurface having patterned hydrophobic and hydrophilic regions

Embodiments also include a kit for storing microfluidic samples. The kitcontains any of the interfaces described above as well as a chipcontaining the array of sample wells. In such a kit, the interface mayfurther contains a reagent for the wells in the selected subset ofsample wells. For example, the reagent may be a dye for staining thesample fluid populated into the subset of wells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a detailed cut away view of a typical nanoliter sample chipaccording to the prior art.

FIG. 2 shows a top plan view of a chip according to FIG. 1 in which thearray of sample wells is grouped into sub-arrays.

FIG. 3 shows various details of channel geometry for use in a fluidaccess structure.

FIG. 4 shows a cross section of a PDMS loader interface according to oneembodiment of the present invention.

FIG. 5 shows a PDMS loader interface having a hard plastic over-layeraccording to one embodiment.

FIG. 6 shows an alternative embodiment of a PDMS loader interface with ahard plastic over-layer.

FIG. 7 shows an embodiment in which a portion of the sample chip isfolded to enable a sub-array to fit into a sample well of a microplatearray.

FIG. 8 shows a mask-based embodiment of an interface loader.

FIG. 9 shows use of contact printing as an interface loader mechanism.

FIG. 10 shows an embodiment in which a porous membrane serves as aninterface loader mechanism.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Various embodiments of the present invention are directed to providingan interface for storing microfluidic samples in an array ofthrough-hole sample wells. A fluid access structure provides a fluidaccess region to a selected subset of sample wells. A fluid introductionmechanism introduces a sample fluid to the fluid access region so thatthe sample wells in the selected subset are populated with the samplefluid without the unselected sample wells being populated with thesample fluid.

A variety of factors affect how specific embodiments are realized. Amongthese is the need for uniformity—the specific process should approachthe uniformity of batch loading techniques, with minimal fluidics errors(e.g., less than 1% of the selected sample wells fail to properly loadthe sample fluid). Also, dead volume (unused sample fluid left in theloading interface) should be minimized to the extent possible; inefficient embodiments, dead volume may be less than 10% of the totalsample fluid volume. In addition, cross-contamination (cross-talk) needsto be avoided between the selected sample wells and the unselectedsample wells.

Other factors that influence specific embodiments include specificdetails of the intended application. For example, whether manual loadingor robotic loading will be used to provide sample fluid to the fluidintroduction mechanism, the sample source structure (e.g., 384-wellmicroplate), and compatibility with other handling procedures such asuse of perfluorinated liquids. Also, the amount of space betweenadjacent sub-arrays affects susceptibility to cross-talk.

After the sample fluid has been loaded into the wells in the subset(sub-array), the loader interface containing the fluid access structureand the fluid introduction mechanism may be removed, for example bypeeling or prying it off the surface of the sample chip. In oneembodiment, the sample chip and loader interface come packaged togetheras a kit in which the loader interface is pre-affixed to the sample chipensuring proper alignment between the two. In some specific embodiments,it may be useful to provide reagents in a dry form on the walls of theinterface loader structures. Structures associated with a givensub-array may have the same reagent or different reagents. The reagentsmay be encapsulated in a gel or wax such as polyethylene glycol (PEG).Par example, a fluorescent dye may be coated on the interior walls of aloader interface so that when a biochemical sample such as nucleicacids, cells, or proteins are added to a given sub-array, they arestained with the dye.

In one specific embodiment, the fluid access structure is adapted forpositioning next to a planar surface of the sample chip to provide thefluid access region, for example, by providing at least one microfluidiccircuit for distributing the sample fluid to the fluid access region.Such a microfluidic circuit may be based on microfluidic channels in thefluid access structure such that the channels overlay and connect theopenings of the subset of sample wells in the fluid access region. Thefluid introduction mechanism may be a port or reservoir that suppliessample fluid to the channels. For example, a pipette or micro-syringemay provide sample fluid to a fluid introduction mechanism such as adocking port that receives the sample fluid. The docking port connectswith the access structure channels that form the fluid access region.The sample fluid in the channels then is populated into the selectedsubset of sample wells in the sample chip. In various embodiments, theremay be one docking port per channel, or a plurality of docking ports perchannel.

The microfluidic channels, while open on the bottom side that faces thesample chip, may be either closed or open on top. Channels that are openon the top have the advantage of being easier to load by hand or with arobotic dispensing station having ordinary precision, since a dropletneed only contact the microfluidic circuit fluid access structure at anyposition on the structure. Open-top structures are typically easilyproduced from rigid materials such as steel, titanium, glass or siliconbut these rigid structures may be expensive as in the case of silicon,or of insufficient flatness and flexibility to provide intimate contactwith the underlying array as in the case of steel A closed-top structuremay be easier to manufacture from elastonmeric materials, but mayrequire the use of ports and docking of dispensers to those ports aswell as regulation of the pressure applied by the dispensers. The fluidaccess structure may be produced from various materials, includingwithout limitation metal, plastic, and glass. In one specificembodiment, silicon was used to fabricate the fluid access structure andwas found to be easy to handle, with good rigidity, but also relativelyfragile, easily breakable, and expensive to produce. One way to benefitfrom the rigidity and open top design of hard materials with theintimate fluidic contact of soft or elastomeric materials is to coat astructure produced with a hard material such as steel with a softmaterial such as PDMS.

Another embodiment may be based on metal such as stainless steel. Steelis easy to handle, inexpensive, and possesses excellent rigidity andstrength. Steel also is hydrophilic, which helps hold the sample fluidin the channels. To avoid cross-talk, a steel fluid access structure mayinclude a hydrophobic monolayer surface coating, such as ofoctadecyltrichlorosilane (OTS). To promote good wetting properties andbiocompatibility of the inside walls of a microfluidic circuit, thesemay be selectively coated with a hydrophilic material. The hydrophilicmaterial may, without limitation, be a deposition of hydrophilic andpreferably biocompatible wax such as polyethylene glycol (PEG), or acovalently linked coating such as a silane bearing PEG moieties.

The channels in a steel fluid access structure can be produced byvarious different methods such as etching or Electrical DischargeMachining (EDM). EDM uses high-energy electric current to melt the basemetal for burr-free machining. Wire EDM can produce intricate patternsand complex shapes with great precision and minimal variation.

FIG. 3A shows some examples of various channel shapes for use in a fluidaccess structure. The chip sample wells 12 are the small holes seen inFIG. 3A. Among the channel shapes are a serpentine geometry 31, anirrigation row geometry 32, and a spiral geometry 33. There may be afluid introduction mechanism such as a docking port and sample reservoirconnected to a point in a given geometry. Then, the sample fluid isdelivered from the fluid introduction mechanism to the microfluidicchannel(s) of the fluid access structure. As the sample fluid travelsdown the channel over the opening of a sample well, it is wicked intothe well by capillary action to fill a volume of the sample well withthe sample fluid.

Depending on the specific channel shape, and other factors such as thegeometry of the sample chip, the width of the fluid access structurechannels needs to be properly dimensioned to be neither too narrow nortoo wide. FIGS. 3B and 3C show cross-sectional views of two differentchannels. FIG. 3B shows a sample loader interface that is 500 μm thickhaving a 140 μm wide channel with perpendicular walls. FIG. 3C shows asample loader interface that is 300 μm thick having hourglass-shapedchannel walls that are 320 μm thick at the center and 450 μm thick atthe surface. In one specific embodiment, the width of the channels maybe the same as the diameter of the sample well openings. In anotherembodiment, the channels are narrower than the diameter of the samplewell openings. In some geometries, thinner channels may be preferred asproviding better sample transfer characteristics, and channels that aretoo wide may have problems filling spontaneously with sample fluid froma pipette, or may not transfer sample fluid efficiently to an adjacentsample chip. In some embodiments, the fluid access structure is the samethickness as the sample chip, so that there is a 1:1 aspect ratiobetween the sample wells and the microcircuit channels, e.g., both thefluid access structure and the sample chip may be 300 μm thick. Also thethicker the fluid access structure is, the greater the undesirable deadvolume of untransferred sample fluid may be. Thicker fluid accessstructures may also be harder to load with sample fluid.

It is important to obtain good planar surface contact between the samplechip and the fluid access structure. Poor contact may result ininconsistent loading and other problems. It may be more or lessdifficult to fabricate some materials in the desired geometries anddimensions with the necessary flatness and rigidity. Moreover, somematerials may be more prone to being deformed when handled. Somematerials may have issues with burs and other fabrication irregularitiesthat may interfere with proper operation.

One means to enhance contact is to apply pressure to press the samplechip and the fluid access structure together, for example by clamping.In some embodiments, magnetic materials may assist in forming propersurface contact between the sample chip and the fluid access structure.Gaskets may also be useful for connecting the chip and the fluid accessstructure. For example, an elastomeric polymer such asPolydimethylsiloxane (PDMS) may be used as a gasket in some embodiments.In other embodiments, a sandwiched layer of PDMS usefully connects theplanar surface of the sample chip and the fluid access structure.

In another embodiment, the sample loader interface itself may be basedon a elastomeric material such as PDMS. That is, the channels of thefluid access structure and the sample receiving port of the fluidintroduction mechanism may be cast in PDMS. PDMS is naturally soft andtacky, and it can cast fine features in the range of 10-50 μm.

FIG. 4 shows a cross section of a PDMS loader interface 40 having aserpentine geometry 31 as seen in FIG. 3A. FIG. 4A shows a cross-sectionof the fluid introduction interface which includes a docking port 41into which a pipette or microsyringe containing the sample fluid may beinserted. At the bottom of the docking port 41 is a sample reservoir 42which holds a volume of sample fluid for delivery into the microfluidicchannels of the fluid access structure. FIG. 4B shows a cross-sectionthrough a microfluidic channel 43 which overlays the openings of thesample wells in the serpentine geometry 31 shown in FIG. 3A. Thus,sample fluid from a pipette or microsyringe in the docking port 41 isdelivered via the sample reservoir 42 to the microfluidic channel 43. Asthe sample fluid travels down the channel 43 and passes over the openingof a sample well, it is wicked into the sample well by capillary actionand the sample well is populated with a volume of the sample fluid. Ifthe sample fluid is provided with too much pressure, some fluid mayescape the reservoir 42 or channel 43 and cause cross-contamination(cross-talk).

A PDMS loader interface can conveniently be produced by casting polymerresin on a mold mask having the desired features and geometry. Forexample, a prototype interface can be produced in PDMS resin by usingstereolithography to convert three-dimensional CAD data into a series ofvery thin slices. A laser-generated ultraviolet light beam traces eachlayer onto the surface of the liquid polymer, forming and hardening eachlayer until the complete, full-size prototype is formed. Anothertechnique for forming a polymer-based loader interface may useultraviolet lithography to develop an SU-8 photo resist structure. Itmay be useful to experimentally vary the ratio of resin base todeveloper, as well as the settling and curing times and temperatures inorder to remove a cast interface from its mold without damage. Ingeneral, slower curing at lower temperature may work better, as highertemperature curing may cause the molded interface to be too brittle.Access ports for the fluid introduction mechanism can be molded in, oradded after molding by boring, laser machining, punching, or drilling ahot needle.

Although the channels of the loader interface need to be hydrophilic inorder to properly transport and deliver the sample fluid, PDMS isnaturally hydrophobic and it needs special treatment to becomehydrophilic. It is known in the art to treat PDMS with plasma gas tochange it from hydrophobic to hydrophilic. One drawback of plasmatreatment is that it has been known to degrade over time to return backto its natural state. Another treatment approach is to deposit ahydrophilic coating on the channel surfaces, such as from a solution ofpolyethylene glycol (PEG). Another possibility is a combined treatmentwith plasma and PEG. By coating the interior surfaces as with PEG, andthen allowing the other surfaces to revert to hydrophobicity or treatingthese surfaces to render them hydrophobic, a selectively coatedelastomer structure results which may be optimal in both ease of loadingand prevention of sample crosstalk.

In some applications, the soft resiliency of PDMS can cause problemswith the fluid introduction mechanism, specifically, the docking portsmay be difficult to use. One solution is to overlay the main PDMSstructure with a layer of hard material such as hard plastic. FIG. 5shows such an embodiment in which a hard plastic over-layer 50 lies ontop of a PDMS loader interface 40 and sample chip 10. The over-layer 50includes an oversize docking port 51 which by virtue of its larger sizeand harder plastic material may act more effectively to receive the endof a pipette or microsyringe delivering the sample fluid.

FIG. 6 shows a further embodiment in which the hard plastic over-layer50 wraps around the sides of the PDMS loader interface 40 and samplechip 10. This configuration can provide added stability and rigidity tothe entire structure and help maintain proper registration (alignment)between the PDMS loader interface 40 and the sample chip 10.

Microfluidic circuits may also be used with other non-through-holemicroarrays including nucleic acid hybridization or protein arrays onglass slides. Microfluidic circuit-based fluid access structures may bevery effective and may avoid many sample transfer problems such assmearing and blotching of sample fluid across the surface of the samplechip in and around the fluid access region. But microcircuits maywastefully retain some of the sample fluid in an unused dead volume.

Another embodiment may be based on a three-dimensional structure havingsub-arrays of sample wells to avoid such dead volume problems. Astructure may be adapted to allow simultaneous access to the benefits ofa high-density nanoliter array format, and the automated liquid-handlingadvantages of commercial microtiter plates. Unlike the two-dimensionalplanar nanoliter sample chip shown in FIG. 1, such embodiments arethree-dimensional with sub-arrays of sample wells connected to eachother by a structure that is above the plane of the sample wells tofacilitate mating with a microtiter plate.

One difficulty in manufacturing such a microtiter-compatible loaderinterface is that techniques for producing the through-hole nanolitersample wells require the substrate to be planar. One approach would beto micromold from a suitable polymer a three-dimensional structurecompatible with a standard size microtiter plate, the micromoldingcreating the desired through-hole nanoliter sample well geometry at thecorrect locations that will be mated with the microtiter plate.Alternatively, an embodiment could be made of multiple components thatrequire assembly in order to generate the required structure for matingwith a microtiter plate.

In another specific embodiment, a planar material such as a metal can beetched using conventional photochemical fabrication methods. Then twoadditional folding steps may be used to produce the requiredthree-dimensional structure. With proper design of the initial planarpart, the final fabricated structure can be made to match with amicrotiter plate so that sub-arrays of sample wells fit inside the wellsof the microtiter plate. Such an embodiment has the advantage of noassembly steps, together with the reliability and precision ofphotochemical etching, and the ease of forming thin sheet metal.

FIG. 7A shows the initial etched planar piece of such a foldable loaderinterface 70. The structure arms in FIG. 7A will ultimately become thefluid introduction mechanism 71 for introducing the sample fluid in themicrotiter plate wells to the fluid access regions 72 that are the nodesin FIG. 7A. The fluid access regions 72 shown in FIG. 7A each have a 5×5sub-array of 25 through-hole nanoliter sample wells 12 for holding thesample fluid from the fluid access regions that are the microtiter platewells. The number of sample wells in each sub-array can be easilychanged changing the size of the node. If the sample wells are etched ata higher density, 1000 or more sample wells per node is possible. In theinterface 70 shown in FIG. 7A, there are 96 nodes (though 384 would beequally easy to manufacture). The work piece shown in FIG. 7A is theinterface 70 after photochemical etching, but before forming. Theoutside frame could be removed before the forming operations, or itcould be left attached and used to handle the final part.

The interface 70 shown in FIG. 7A can be finished by using two formingdies that are designed so that they each act on only one direction ofthe work piece. The first forming operation would then bend the all ofthe material in one direction—for example, all rows—and leave thematerial connections on the columns undisturbed. An example of a portionof the resulting work piece is shown in close-up in FIG. 7B. The finalforming operation would be orthogonal to the first to then shape all ofthe columns. A portion of the final formed interface 70 would be asshown in the close-up in FIG. 7C. The final formed interface 70structure can then match the top of a standard 96-well microtiter sampleplate. This allows the nanoliter-size sample wells in the sub-arrays ofeach fluid access region 72 to be inserted and withdrawn numerous timesinto the wells of a microtiter plate (as well as various other liquidreceptacles) in order to perform various steps in one or more assayoperations.

To use such a three-dimensional loader interface, reagents can bepre-formatted into the sample wells of the unformed planar work piece,for example, using pin transfer technology. Alternatively, the interface70 may first be formed into its final shape, and then inverted to allowreagents to be transferred into the sample wells by pin transfer. Thetransferred reagents may be fixed onto the walls of the sample wells bydrying, and then released upon dipping the interface 70 into amicroplate with sample fluid in its wells. In the specific case of PCR,thermal cycling would follow. Wash operations may also be performed bydipping the assembly into a trough or a microplate as for an ELISA.After performing analytical reactions, the plate may be imaged with alaser scanner or high resolution CCD-based system in any availablereadout mode.

There are also a variety of other approaches to provide a sample loaderinterface to a sample chip. FIG. 8 shows an embodiment in which thefluid access structure and fluid introduction mechanism are integratedtogether into a mask overlay. A resilient material such as PDMS orsilicone divides the surface of the sample chip by creating fluidbarriers between sub-arrays. In a mask-type application, it may beuseful to place the sample chip onto a hydrophobic surface to preventthe sample fluid from spreading across the bottom of the chip.Alternatively or in addition, various embodiments may employ a mask onthe top of the sample chip and a similar corresponding mask on thebottom of the sample chip to avoid cross-talk. Chips intended for usewith sub-array masks may also have ridges and other surface featuressuch as spacing arrangements to aid with registration of the mask withthe sample chip.

It may also be useful to blot the surface of the chip after addingsample fluid to one of the sub-arrays. For example, a serpentine loadercircuit such as shown in FIG. 3A may be laid over the sub-array filledusing the mask in order to blot up excess sample fluid. Mask-basedembodiments may have difficulties with blotching of the sample fluidleading to cross-talk. The mask is typically removed from the arrayafter blotting and prior to use.

Masking performance may also be improved by using a centrifuge loadingtechnique. In addition or alternatively, sample fluid may be introducedinto a masked sub-array by a variety of means including withoutlimitation use of a swab, brush, pad, or sponge.

FIG. 9 shows another embodiment in which the sample fluid is transferredto a selected sub-array of sample wells by printing. As shown in FIG.9A, a hydrophilic island 91 in a background of hydrophobic areas on aprinting plate 90 is loaded with sample fluid 92, for example by use ofa pipette. The printing plate 90 is then pressed down into contact withthe openings of a selected set of sample wells 12 in a sub-array onsample chip 10. Sample liquid is then wicked by capillary action intothe selected sample wells 12 and the printing plate 90 is lifted off ofthe sample chip 10. As with mask-based embodiments, it may then beuseful to blot the surface of the sub-array, for example with aserpentine circuit interface, to remove any excess sample liquid fromthe surface of the sub-array. In some printing-based embodiments, it maybe difficult to prevent spreading of the printed sample fluid whichcould lead to cross-talk. Other potential problems include difficultiesaligning the printing plate 90 with the sample chip 10, the multi-stepnature of the printing process, and general messiness in the process.

Transferring sample fluid by dragging a hanging drop across the surfaceopenings of selected sample wells may be useful either in combinationwith various of the above embodiments, or on its own. A pipetter,capillary tube, microsyringe, cannula, pin, or the like may be used todispense and drag droplets across selected sub-arrays. This may be aidedby use of a liquid handling station such as a reformatter, BioMek™(marketed by Beckman Coulter of Fullerton, Calif.), or other commercialsystem. For example, a sample chip may be positioned beneath an array ofhanging drops in a jig that confines the movement of the sample chipwithin a defined region in a plane, such as a 45 mm square. The samplechip is then moved beneath the hanging drops to distribute sample fluidinto the selected sample wells. Transferring sample fluid to a nanolitersample chip by banging drops is described in U.S. patent applicationSer. No. 09/850,123, filed May 7, 2001, and incorporated herein byreference.

Other non-contact techniques for transferring sample fluid to selectedsample wells may be useful either in combination with various of theabove embodiments, or on its own. For example, focused non-contact dropdispensing (drop spraying) may be used to direct sample liquid intosample wells. The hanging droplet may be dragged to a dedicated orunused area of the array or sub-array to facilitate removal of excesssample. A non-contact dispensing system is available from LabCyte ofSunnyvale, Calif.

FIG. 10 shows an embodiment in which a porous membrane serves as aninterface loader mechanism. In the embodiment shown, microporousmembrane 100 has internal unidirectional pores having hydrophilicsurfaces. The outer surfaces 101 of the membrane are patterned to begenerally hydrophobic with hydrophilic areas that correspond to theopenings of the selected sample wells 12 in the sub-array on sample chip10.

Such a porous membrane 100 may be attached to the ample chip 10 by avariety of different means, for example, by a wax. The specificattachment mechanism should prevent cross-talk of sample fluid beyondthe sub-array defined by the membrane 100, while allowing for easyremoval of the membrane after sample fluid has been added to the samplewells 12 in the sub-array. In addition or alternatively, the membrane100 can be placed in a flexible frame that fits over the sample chip 10to ensure proper alignment with the sub-array sample wells 12 into whichsample fluid is to be dispensed.

As shown in FIG. 10A, membrane 100 is laid on top of the sample chip 10such that the hydrophobic surface 40 of the chip is in contact with thepatterned hydrophobic outer surface 101 of the membrane. Sample fluid isdispensed onto the top of the membrane 100 and wicked into the interiorpores of the membrane by capillary action. As additional sample fluid isdispensed on top of the membrane, the liquid moves through the interiorpores of the membrane and cannot pass through the hydrophobic regions ofthe outer surface 101 of the membrane (which additionally lies againstcorresponding portions of the hydrophobic surface 40 of the sample chip10). But the sample fluid can and does pass through the hydrophilicportions of the outer surface 101, which are patterned to correspond tothe openings of the selected sample wells 12 in the sub-array. As thesample fluid starts to emerge from hydrophilic regions in the bottom ofthe membrane 100, the liquid comes into contact with and wets thehydrophilic surface of the inside walls of the sample wells 12. Thiscauses the sample fluid to be drawn out of the membrane 100 by capillaryaction and into the interior volumes of the sample wells 12 until theyare filled.

After sufficient time, the membrane 100 can be peeled away from thesample chip 10 as shown in FIG. 10B such that the shear force breaks thefluid bridge between the sample fluid remaining in the membrane 100 andthe sample fluid in the sample wells 12. The membrane 100 can then bediscarded and the sample chip 10 is ready for use.

The total volume of sample fluid dispensed onto the top of the membrane100 should be controlled in order to avoid wetting of the outer surface40 of the sample chip 10. If the volume of sample fluid that isdispensed exceeds the combined volume of the membrane 100 and theselected sample wells 12, then the outer surface 40 of the sample chip10 will most likely wet. Dispensing less than this critical volumeensures that the excess fluid remains within the membrane 100 as it isremoved from the sample chip 10. Furthermore, the shear force applied tothe liquid bridge as the membrane 100 is peeled off minimizes thepossibility of chip surface wetting.

Assuming that the dispensing area of the membrane 100 is fixed by thenumber of sample wells 12 to be addressed in the selected sub-array,dead volumes can be minimized by controlling the thickness of themembrane 100. For example, a 300 μm² 8×8 sub-array of 64 sample wellshaving individual storage volumes of 25 nanoliters channels has a totalcombined volume of 1.6 microliters. If the membrane is 250 μm thick,then approximately 3 microliters of sample fluid needs to be loaded intothe membrane in order to deliver 1.6 microliters to the sub-array. Thismeans approximately 50% of the sample fluid is wasted in dead volume(1.4 microliters).

Membrane-based interface loaders accommodate different automatic orhand-dispensing mechanisms including pipettes or syringes with cannula.The membrane can be partitioned in various ways to ensure that samplefluid passes only into a given selected sub-array of sample wells. Forexample, a large number of unidirectional pores may connect the upperand lower surfaces of the membrane so that sample fluid is transferredsubstantially perpendicularly to these bounding surfaces, ensuring thatsample fluid goes Sonly to sample wells directly beneath the dispenser.

Alternatively, the membrane may use blocking of pores in a pattern thatis the negative of the sample fluid distribution pattern applied to thesample chip. For example, all the pores in the membrane could be blockedby a hydrophobic epoxy except for a small area into which the samplefluid is dispensed. This embodiment does not necessarily requireunidirectional pores.

There are several membrane attributes that would be desirable. Theseinclude:

-   -   High porosity to ensure transfer to all the sample wells of the        sub-array    -   Thick and durable enough to be applied and removed easily    -   Blotters should not tear when wet and should absorb so that        excess sample fluid is contained and does not cross into another        sub-array.    -   Unidirectional pores to ensure directional flow of sample fluid        from one side of the membrane to the other and into the sample        wells of the sub-array.    -   Patterns of hydrophobic and hydrophobic surface coatings to        facilitate the movement of sample fluid through the membrane        into the sample wells of the sub-array.    -   Segmentation of the membrane to ensure sample fluid applied to        the upper surface of the membrane flows through to a selected        subset of underlying sample wells.

One specific embodiment uses track-etched polyester or polycarbonate.Such an embodiment may have internal pores of a defined size range anddensity, but membrane porosity may be relatively low (5-20%). Such amembrane may be relatively thin, for example, 10-20 μm, and therefore,may be difficult to handle.

Another specific embodiment uses cast membranes-mixtures of celluloseesters (cellulose nitrate and cellulose acetate) which are formed into afibrous network similar to paper. These membranes have an open cellstructure with high porosity (70-80%) and have a broad pore sizedistribution (e.g. 0.22-5.0 μm) which may enhance fluid passage anddistribution to the selected sample wells. These membranes tend to bethicker than track-etched (100-200 μm), which could improve handlingcharacteristics.

Another embodiment uses an Anopore™-aluminum oxide membrane with arelatively high porosity (40-50%) having a honeycomb structure thatensures proper distribution across the sub-array. In this membrane, thepore sizes (20-200 nanometers) may be much smaller than the openings ofthe sample wells.

Yet another embodiment uses a membrane made of paper or glassmicrofiber. Such materials come in different grades with differentspeeds of filtration. Paper filters also come strengthened with resin toenhance durability.

An additional benefit of a membrane loader interface is that it iswell-suited for blotting away from the surface of the sample chip anyexcess sample fluid. But this blotting action should be controlled toprevent the membrane material from pulling sample fluid back out of theloaded sample wells in the sub-array when the membrane is removed. Inother embodiments, the membrane may be used as a blotting mechanism toremove excess sample fluid from the surface of the sample chip after thesample wells in the selected sub-array have been loaded by anothermechanism, for example, by a microfluidic circuit arrangement.

Although various exemplary embodiments of the invention have beendisclosed, it should be apparent to those skilled in the art thatvarious changes and modifications can be made which will achieve some ofthe advantages of the invention without departing from the true scope ofthe invention.

1. A method of storing microfluidic samples, the method comprising:providing a chip having an array of sample wells for storingmicrofluidic samples; providing a fluid access region to a selectedsubset of sample wells; and introducing a sample fluid to the fluidaccess region so that the sample wells in the selected subset arepopulated with the sample fluid without the unselected sample wellsbeing populated with the sample fluid. 2-48. (canceled)